During 2012, we performed laboratory experiments based on previous observations made during our field work in Montana and California. Our investigations of tick-borne relapsing fever in western Montana produced our finding of both genomic groups of Borrelia hermsii in ticks and small mammals at Flathead Lake. Also, one patient that was infected on Wild Horse Island was dually infected with Borrelia hermsii spirochetes belonging to different genomic groups. We also found that two patients that had slept together in the same bed were each infected with spirochetes belonging to different genomic groups. Therefore, we became quite interested in determining how spirochetes in genetically different clones were maintained at one endemic focus. One question to address was how the tick vector might handle dual or super-infections with different strains of B. hermsii. To test the vector competence of Ornithodoros hermsi with distinct Borrelia hermsii genotypes, we adapted an artificial infection method that we developed previously for the Lyme disease spirochete Borrelia burgdorferi and its ixodid hard tick vector Ixodes scapularis. The method involves the submersion of ticks into a liquid culture suspension of spirochetes. Here we adapted the method to infect Ornithodoros hermsi ticks with various isolates of the relapsing fever spirochete Borrelia hermsii. While we were interested in determining if we could infect soft ticks by immersion, we also were stimulated to attempt dual infections because of our findings while studing relapsing fever in Montana as described above and in another annual report. Ornithodoros hermsi larvae were immersed in suspensions of multiple genomic group I and genomic group II isolates to test vector competence with the two genomic groups. We pretreated larvae at 42% relative humidity for 48 hours as this drier pre-immersion exposure increased the amount of the liquid suspension of spirochetes the tick larvae imbibed. After immersion, the tick larvae were allowed to recover for two weeks before feeding them on mice. After the immersed larvae were fed on mice and allowed to molt to the nymphal stage, they were fed again on mice to determine if the ticks were capable of transmitting spirochetes. Regardless of the strain of spirochete of Borrelia hermsii used, the larvae became infected by immersion, maintained the infection through the molt, and transmitted spirochetes when they fed as nymphs. Larvae were also immersed with mixtures of spirochetes that comprised different strains, and again the immersed ticks became infected and subsequently transmitted spirochetes. For those ticks exposed to a mixture of genomic types, these ticks transmitted both types of spirochetes when they fed on mice. These results suggest that ticks in an endemic focus may be able to become dually infected and co-transmit multiple genetic types of Borrelia hermsii during a single feeding. In another series of on going experiments, we have investigated the prevalence of transovarial transmission of Borrelia hermsii by female ticks. This mode of vertical transmission of spirochetes, from infected females to her offspring, has been proposed to be a rare event for Ornithodoros hermsi ticks infected with B. hermsii. During our investigations of relapsing fever at Mt. Wilson near Los Angeles, California, we collected infected nymphal ticks that became adults in the laboratory. The strain of Borrelia hermsii in these ticks is the most virulent type of this bacterium we have observed in our 25 years of working with this species of spirochete. Therefore, we have begun a study to follow the offspring of ticks that originated from the single female that developed from one of the field-collected nymphs. To our surprise, transovarial transmission is prevalent in this colony of ticks, with nearly 100% of the progeny becoming infected. These results are based on microscopic examination of larvae, PCR analysis of nymphs, and numerous feedings of single and multiple ticks with transmission to mice. As well, the transovarially-infected larvae maintain the infection transstadially through the multiple nymphal stages, to adults. These F-1 generation females are also infected, transmit spirochetes, and produce infected progeny. Ticks in this colony are still being tracked, but our results so far demonstrate conclusively that for some strains of Borrelia hermsii, the bacteria are transmitted transovarially by infected females at a very high frequency. Another area of new research involves the ability of Oornithodoros hermsi to feed upon other individual ticks, which may result in a novel mechanism for the horizontal transmission of Borrelia hermsii in nature. This phenomenon is called hyperparasitism, and so far we have found this behavior to occur quite frequently when unfed ticks are placed together with ticks that recently fed on mice. Male ticks hyperparasitize female ticks at a higher frequency than do the nymphs and females. Also, when unfed ticks attach to ticks that had recently acquired spirochetes when feeding on infected mice, they are capable of acquiring spirochetes and subsequently transmitting them to naive mice during their next bloodmeal. Thus, this phenomenon of spirochete transmission via hyperparasitism provides another mechanism for the maintenance of spirochetes in nature that does not require the vertebrate host. This mechanism of transmission will be investigated further during the coming year.